EP2479268A1 - Procédé de dissociation et agent de dissociation pour avidine et biotine - Google Patents

Procédé de dissociation et agent de dissociation pour avidine et biotine Download PDF

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Publication number: EP2479268A1
Authority: EP; European Patent Office
Prior art keywords: biotin; avidin; derivative; bound; water
Prior art date: 2009-09-17
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.): Granted

Application number

EP10817228A

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German (de)

English (en)

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EP2479268B1 (fr

EP2479268A4 (fr

Inventor

Satoshi Katayose

Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)

JSR Corp

Original Assignee

JSR Corp

Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)

2009-09-17

Filing date

2010-09-16

Publication date

2012-07-25

2010-09-16 Application filed by JSR Corp filed Critical JSR Corp

2012-07-25 Publication of EP2479268A1 publication Critical patent/EP2479268A1/fr

2014-03-12 Publication of EP2479268A4 publication Critical patent/EP2479268A4/fr

2020-11-04 Application granted granted Critical

2020-11-04 Publication of EP2479268B1 publication Critical patent/EP2479268B1/fr

Status Active legal-status Critical Current

2030-09-16 Anticipated expiration legal-status Critical

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0 C*C(C)NC(NC)=O Chemical compound C*C(C)NC(NC)=O 0.000 description 1

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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes

Definitions

the present invention relates to a method for dissociating avidin or streptavidin from a biotin derivative, and a dissociation agent for use in the method.
(strept) avidin has highly specific affinity with biotin or any derivatives thereof. When the both are mixed with each other under a physiological condition, they are easily bound to form a complex (the complex between (strept)avidin and biotin or between (strept) avidin and the biotin derivative are generally called as "ABC complex"). This property is utilized for isolation of a target substance or a target material such as cells (Patent Literature 1).
a carrier for capturing a target substance may be prepared and the target substance may be captured by use of this carrier, for example, a biotin-labeled probe molecule (such as an antibody having binding affinity for the target substance), to which biotin is beforehand bound , are mixed with an insoluble carrier to which (strept)avidin is immobilized under a physiological condition, to form an ABC complex. Thereafter, if the ABC complex or the binding between the probe molecule and the target substance can be cleaved, the target substance can be isolated (Patent Literature 1).
a biotin-labeled probe molecule such as an antibody having binding affinity for the target substance
a target to be isolated is a protein, cells, or other target substance that is easily damaged by a severe pH, heat or salt concentration, or physical shear force, it is generally difficult to isolate the target substance wherein the physiological activity thereof is maintained. Therefore, methods for easily isolating a target substance, for example, a method using a biotin derivative which binds to (strept) avidin with lower affinity than biotin (Patent Literature 2); and a method using an avidin variant lower in affinity with biotin than avidin (Patent Literature 3), are proposed.
Patent Literature 2 a method in which, instead of biotin, desthiobiotin or some other biotin derivative which binds to (strept)avidin with lower affinity than biotin (Patent Literature 2) is used to prepare a capturing carrier as described above, a target substance is captured thereby, and then a large amount of biotin is added thereto for cleaving the ABC complex to isolate the target substance.
the biotin is thought to function as a competitive inhibitor for bonding between desthiobiotin and (strept)avidin.
this method in which biotin is used as competitive inhibitor it would be difficult to efficiently cleave the ABC complex.
the target substance would be difficult to be efficiently isolated.
the present invention provides a method for dissociating (strept) avidin efficiently from a biotin derivative with lower affinity than biotin, as mentioned above, under a mild condition in a short period, and a dissociation agent for use in the method.
the inventor has made various investigations to find out a means for dissociating (strept)avidin efficiently from a biotin derivative with lower affinity than biotin, as mentioned above.
the inventor has unexpectedly found out that by use of a water-soluble polymer to which biotin or the like is bound as a dissociation agent, (strept)avidin is efficiently dissociated from the biotin derivative with lower affinity than biotin under a mild condition.
the invention has been provided.
the invention provides a method for dissociating avidin or streptavidin from a biotin derivative (1) which binds to avidin with lower affinity than biotin, comprising the step of mixing a combination of avidin with the biotin derivative (1), or a combination of streptavidin with the biotin derivative (1) (hereinafter the combination being referred to as "avidin-biotin complex”) with a water-soluble polymer to which biotin or a derivative (2) thereof is bound (hereinafter the polymer being referred to as "dissociation agent").
the invention also provides a method for isolating a target substance, comprising:
the invention provides a dissociation agent which is used for dissociating avidin or streptavidin from a biotin derivative (1), and contains a water-soluble polymer to which biotin or a derivative (2) thereof is bound.
the complex between (strept)avidin and the biotin derivative (1) can be efficiently cleaved under a mild condition in a short period to dissociate (strept) avidin and the biotin derivative (1) from each other.
this method is applied to the isolation of a target substance, the obtained target substance bound to the capturing carrier can easily be isolated.
the target substance is a substance with lower stability against temperature condition, pH condition or some other condition, such as a protein or one or more cells, the use of the invention makes it possible to isolate the target substance in which the activity thereof is maintained.
the method according to the embodiment of the invention for dissociating (strept)avidin from a biotin derivative (1) comprises the step of mixing an avidin-biotin complex with a water-soluble polymer to which biotin or a derivative (2) thereof is bound.
the dissociating method of the invention is carried out in a solvent comprising, mainly, water.
the avidin-biotin complex exists in the solvent as dissolved form.
the avidin-biotin complex may be dispersed, together with the carrier, into the solvent.
the avidin-biotin complex used in the invention is a combination of avidin or streptavidin with a biotin derivative (3) which binds to avidin with lower affinity than biotin (biotin is a compound represented by the below formula (3)).
biotin is a compound represented by the below formula (3).
Avidin or streptavidin used herein may be a commercially available product. They may be a compound isolated from the nature world and then purified, or a compound produced artificially by genetic engineering technique, or an avidin or streptavidin derivative that is modified or varied within the degree that the binding capacity thereof to biotin is not lost.
the avidin or streptavidin derivative examples include chemically modified (for example, succinylated) avidin or streptavidin subjected, and an avidin or streptavidin monomer (that is, the avidin or streptavidin usually forms a tetramer thereof).
the biotin derivative (1) is not particularly limited as far as the derivative is a biotin derivative which binds to avidin with lower affinity than biotin. According to the lower binding affinity to avidin of the biotin derivative (1) than biotin, when the avidin-biotin complex thereof is mixed with a water-soluble polymer to which biotin or a derivative (2) thereof is bound, (strept) avidin and the biotin derivative (1) are effectively dissociated from each other.
biotin derivative (1) examples include derivatives having a cyclic moiety of biotin different from that of biotin, such as desthiobiotin (a compound represented by the following formula (4)), 2-iminobiotin (a compound represented by the following formula (5)) and 3,4-diaminobiotin (a compound represented by the following formula (6)) and chemically modified products of these compounds:
(any compound) lower binding affinity with avidin than biotin means that the binding constant thereof (with avidin) is one order or less smaller than that of biotin with avidin (10 15 M).
the binding constant with avidin of each of the compounds represented by the formulae (4) to (6), respectively, is from 10 6 to 10 13 M.
the avidin-biotin complex used in the invention may be immobilized through (strept)avidin to an insoluble carrier.
the avidin-biotin complex When the avidin-biotin complex is immobilized to the insoluble carrier, the avidin-biotin complex can easily be isolated from a dispersion liquid containing the avidin-biotin complex by centrifugation, filtration or other methods.
the form or shape of the insoluble carrier used for immobilization of (strept) avidin is not particularly limited. Examples of the carrier include organic or inorganic particles, a tabular substrate, a substrate having fine channels, and a micro-well plate.
the particles are preferably magnetic particles since the particles can be isolated by magnetism.
examples of a commercially available product of the magnetic particles include Dynabeads M-450 Tosylactivated (manufactured by Invitrogen (Co.)) and Magnosphere MS300/Carboxyl (manufactured by JSR Corp.).
the magnetic particles are preferably produced by a known method (for example, JP-B-H05-010808 , or JP-A-2007-288133 ), and the non-specific adsorption of a protein, cells and others can be decreased.
the method for immobilizing (strept)avidin to the insoluble carrier is not particularly limited, and may be publicly known methods (see, for example, JP-A-2001-158800 ).
(strept)avidin can be immobilized to the insoluble carrier by formation of amide bonds through reaction of amino groups in molecules of (strept) avidin with the carboxyl groups on the carrier surface in the presence of a dehydration condensing agent such as water-soluble carbodiimide.
a dehydration condensing agent such as water-soluble carbodiimide.
the carboxyl groups on the carrier may be reacted with the dehydration condensing agent in advance, and then avidin or streptavidin may be added thereto, to complete the reaction.
the avidin-biotin complex used in the invention may be a combination of the following (A) and (B) (hereinafter the combination being referred to as "capturing carrier”):
the species of the probe molecules is not particularly limited, and examples thereof include an antibody against a target substance, proteins such as lectin and enzymes, and saccharides.
the probe molecules have a binding property to a target substance specifically, and are bound to the biotin derivative (1).
the probe molecules may be bound to the biotin derivative (1) directly or through a spacer.
the biotin derivative (1) immobilized to the probe molecules is a biotin derivative (1) showing lower affinity than that of biotin for avidin, this biotin derivative having been described in the above section regarding the avidin-biotin complex.
the method for bonding the biotin derivative (1) to the probe molecules is not particularly limited, and may be, for example, similar to the above-mentioned method for immobilizing (strept) avidin to the surface of a carrier.
a carboxyl group in biotin molecule may be reacted with amino groups in the probe molecules, in the presence of a dehydration condensing agent such as water-soluble carbodiimide, thereby amide bond is formed, to obtain biotin-bound probe molecules.
FIG. 1 shows a conceptual view of the capturing carrier bound to the target substance.
the target substance is not particularly limited, and examples thereof include cells (for example, various cells of bacteria, eumycetes, animals orplants), viruses, proteins (for example, various antigens, antibodies or enzymes), nucleic acids such as single-stranded or double-stranded DNA or RNA, steroid lipids such as estrogen, glycolipids, and polysaccharides.
cells for example, various cells of bacteria, eumycetes, animals orplants
viruses proteins (for example, various antigens, antibodies or enzymes)
nucleic acids such as single-stranded or double-stranded DNA or RNA
steroid lipids such as estrogen, glycolipids, and polysaccharides.
target cells include normal cells (for example, stem cells such as hematopoietic stem cells, blood cells such as white blood cells), cancer cells (for example, circulating cancer cells in peripheral blood, which originate from mammary cancer, lung cancer, or some other, exfoliative cancer cells present in feces, which originate from colon cancer or some other, cancer cells in menstrual blood, which originate from uterine cancer, and cancer cells present in urine, which originate from bladder cancer).
normal cells for example, stem cells such as hematopoietic stem cells, blood cells such as white blood cells
cancer cells for example, circulating cancer cells in peripheral blood, which originate from mammary cancer, lung cancer, or some other, exfoliative cancer cells present in feces, which originate from colon cancer or some other, cancer cells in menstrual blood, which originate from uterine cancer, and cancer cells present in urine, which originate from bladder cancer.
the target substance can be efficiently isolated by the method for isolating target substance according to the invention.
an avidin-biotin complex is mixed with a water-soluble polymer to which biotin or a derivative (2) thereof is bound (hereinafter the polymer being referred to as "dissociation agent"), to cleave the binding between (strept)avidin and the biotin derivative (1) to dissociate the (strept)avidin from the biotin derivative (1).
dissociation agent a water-soluble polymer to which biotin or a derivative (2) thereof is bound
the dissociation agent is most preferably a water-soluble polymer to which biotin is bound.
biotin derivative (2) that may be used in the dissociation agent include derivatives having the same cyclic moiety as biotin, such as biocytin (biotin-E-N-lysine: a compound represented by a formula (7) illustrated below), biotin-N-hydroxysuccinic imide ester (a compound represented by a formula (8) illustrated below), and a compound represented by a formula (9) illustrated below (biotinyl-3,6,9-trioxaundecanediamine); or derivatives each having a cyclic moiety different from that of biotin, these derivatives having been described in the above section regarding the avidin-biotin derivative.
biocytin biotin-E-N-lysine: a compound represented by a formula (7) illustrated below
biotin-N-hydroxysuccinic imide ester a compound represented by a formula (8) illustrated below
the dissociation agent may be added to a liquid sample containing an avidin-biotin complex under a physiological condition, and then they are mixed with each other.
the physiological condition is preferably the following condition: a temperature of 20 to 40°C, a pressure of about 1 atm., and a pH of 5 to 9.
the (strept)avidin can be dissociated efficiently from the biotin derivative (1) by the use of the dissociation agent of the subj ect invention.
the dissociation agent of the subj ect invention By addition of the dissociation agent to an aqueous sample which contains an avidin-biotin complex, avidin or the derivative (2) thereof contained in the dissociation agent competes with the biotin derivative (1) bound to (strept) avidin, to dissociate (strept) avidin from the biotin derivative (1).
the dissociation agent in which the biotin or the derivative (2) thereof is bound to the water-soluble polymer, can dissociate (strept)avidin more efficiently from the biotin derivative (1) than using free biotin or the derivative (2) thereof which is not bound to any water-soluble polymer or the like, although the reason is not clear.
the water-soluble polymer is not particularly limited as far as the polymer is a water-soluble polymer and can be bound to biotin or the derivative (2) thereof.
the polymer is preferably a water-soluble polymer having an amino group, a sulfhydryl group, a carboxyl group, or some other functional group because the water-soluble polymer can be bound to biotin through a carboxyl group in the biotin molecule.
Examples thereof include water-soluble proteins, polysaccharides, and organic synthetic polymers having reactive functional groups such as amino group.
Preferred examples of the water-soluble proteins include BSA (bovine serum albumin), and HAS (human serum albumin).
the polysaccharides include CMC (carboxylmethylcellulose), and chitosan.
Examples of the organic synthetic polymers having a reactive functional group include PAA (polyacrylic acid), polyallylamine, and other synthetic polymers; and polyamino acids such as polylysine, polyaspartic acid.
water-soluble means a property that a substance can be dissolved in solvent to give a 1 mg/mL solution without generation of white turbidness.
the molecular weight of the biotin-bound water-soluble polymer is not particularly limited, and is preferably from 1,000 to 1,000,000. When the molecular weight of the biotin-bound water-soluble polymer is within this range, the avidin-biotin complex can be efficiently dissociated.
the molecular weight of the biotin-bound water-soluble polymer may be measured by, for example, gel permeation chromatography.
the average molecular number of the biotin or the derivative (2) thereof bound to individual molecules of the water-soluble polymer is preferably as large as possible as far as the water-solubility is not lost.
the average number is preferably from 2 to 2,000, more preferably from 5 to 1,000, even more preferably from 10 to 100.
the average molecular number of the biotin or the derivative (2) thereof bound to individual molecules of the water-soluble polymer may be measured by a method described in Examples described below (HABA method).
the method for binding the biotin or the derivative (2) thereof to the water-soluble polymer is, for example, a method of reaction of biotin or the derivative (2) thereof with a water-soluble polymer having at least one of a carboxyl group and an amino group.
the method may be a method of dehydration-condensation of the amino group in the water-soluble polymer with the carboxyl group of biotin.
the method may be a method of dehydration-condensation of the carboxyl group in the water-soluble polymer with the amino group of the amino-group-having biotin derivative.
the method of the invention for isolating a target substance, includes: the step (a) of mixing a capturing carrier with the target substance in an aqueous solvent to obtain a liquid mixture 1, the step (b) of mixing the liquid mixture 1 with a water-soluble polymer to which biotin or a derivative (2) thereof is bound, to obtain a liquid mixture 2, and the step (c) of isolating the target substance from the liquid mixture 2.
the capturing carrier is:
This step is a step of binding a capturing carrier to a target substance.
a capturing carrier is mixed with a target substance, to bind the probe molecules contained in the capturing carrier specifically to the target substance or to molecules contained in the target substance and having affinity for the probe molecules.
the capturing carrier and the target substance are as described above.
the aqueous solvent is preferably a solvent having a pH of 5 to 9 containing water as a major medium, and is more preferably a buffer solution such as a phosphoric acid buffered solution or a Tris buffered solution.
the capturing carrier In the case of mixing the capturing carrier with the target substance, it is preferred to mix the capturing carrier dispersed in the aqueous solvent with the target substance dissolved or dispersed in the same or different aqueous solvent. After the mixing, it is preferred to stir the mixture at 10 to 40°C for 1 to 60 minutes. By the stirring under such a condition, the capturing carrier and the target substance are efficiently bound to each other.
the amount of the mixed capturing carrier may be appropriately adjusted in accordance with the amount of the target substance contained in the sample.
This step is a step of adding a dissociation agent to cleave the binding between (strept)avidin in the capturing carrier and the biotin derivative (1), for dissociating the (strept) avidin and the biotin derivative (1) from each other.
a dissociation agent to cleave the binding between (strept)avidin in the capturing carrier and the biotin derivative (1), for dissociating the (strept) avidin and the biotin derivative (1) from each other.
This step may include a step of isolating the capturing carrier from the liquid mixture 2.
the capturing carrier contains magnetic particles, the capturing carrier can easily be isolated by use of a magnetic stand.
the present step is a step of isolating the target substance contained in the liquid mixture 2.
the target substance is isolated from the insoluble carrier and the like.
the step may be carried out by any kinds of method.
the capturing carrier contains magnetic particles
the capturing carrier can easily be isolated by use of a magnetic stand.
the method used in the step may be, for example, a method of centrifugation of the dispersion under such a centrifuge condition in which the target substance does not precipitate, a method of filtration of the dispersion through a filter having a pore diameter in the degree that the target substance is not captured.
reaction liquid was washed with 0.05%-Tween-20-containing PBS 3 times to remove unreacted desthiobiotin-bound probe molecules, to yield an avidin-biotin complex (capturing carrier) in which the desthiobiotin-bound probe molecules were bound to the streptavidin magnetic particles.
BSA-Biotin 6 a dissociation agent in which six (6) in average of biotin were bound to one molecule of BSA
BSA-Biotin 10 a dissociation agent in which ten (10) in average of biotin were bound to one molecule of BSA
the quantity of biotin bound to each of BSAs was determined by an HABA method according to Green et al. (4-hydroxyazobenzene-2-carboxylic acid, N. M. Green, Methods in Enzymology, vol. 18, pp. 418-424, 1970 ).
HT-29 cells target cells
2-mM-EDTA-containing PBS 2-mM-EDTA-containing PBS
the number of cells was determined by measuring the quantity of genome DNA according to the quantitative PCR method.
the number of the cells bound to the isolated capturing carrier was measured, and the number was 8,500, which was 85% of the number of the cells used for the Example.
250 ⁇ L of 0.2% by mass solution of the BSA-Biotin 6 in PBS yielded in Preparation Example 3 was added to the capturing carrier bound to the target cells, and mildly mixed at room temperature for 20 minutes. Thereafter, a magnetic stand was used to isolate the particles, thereby the HT-29 cells dissociated in the supernatant were collected. The cells were observed using an optical microscope. As a result, the form of the cells in the supernatant was maintained.
the efficiency of dissociation of the target cells is shown in Table 1.
the item "biotin concentration in dissociation solution” means a value of the percentage by mass of biotin calculated from the concentration of the dissociation agent and the degree of biotinilation of the dissociation agent.
the dissociation efficiency was obtained in the same way as in Example 1, except that a dissociation agent shown in Table 1 was used instead of the BSA-Biotin 6.
the dissociation efficiency was obtained in the same way as in Example 1, except that a 10 mM solution of biotin in PBS was used instead of the 0.2%-by-mass solution of BSA-Biotin 6 in PBS.
Examples 1 to 4 in which the biotin-bound water-soluble polymer was used as the dissociation agent, can dissociate (strept)avidin from the biotin derivative with a very high efficiency to isolate the target cells although the biotin concentration in the dissociation solution was very small.

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EP10817228.9A 2009-09-17 2010-09-16 Procédé de dissociation et agent de dissociation pour avidine et dérivés de biotine Active EP2479268B1 (fr)

Applications Claiming Priority (2)

Application Number	Priority Date	Filing Date	Title
JP2009215732		2009-09-17
PCT/JP2010/066005 WO2011034115A1 (fr)	2009-09-17	2010-09-16	Procédé de dissociation et agent de dissociation pour avidine et biotine

Publications (3)

Publication Number	Publication Date
EP2479268A1 true EP2479268A1 (fr)	2012-07-25
EP2479268A4 EP2479268A4 (fr)	2014-03-12
EP2479268B1 EP2479268B1 (fr)	2020-11-04

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EP10817228.9A Active EP2479268B1 (fr)	2009-09-17	2010-09-16	Procédé de dissociation et agent de dissociation pour avidine et dérivés de biotine

Country Status (5)

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US (1)	US8685655B2 (fr)
EP (1)	EP2479268B1 (fr)
JP (1)	JP5686098B2 (fr)
CN (1)	CN102575244B (fr)
WO (1)	WO2011034115A1 (fr)

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- 2010-09-16 JP JP2011531955A patent/JP5686098B2/ja active Active
- 2010-09-16 EP EP10817228.9A patent/EP2479268B1/fr active Active
- 2010-09-16 CN CN201080040460.7A patent/CN102575244B/zh active Active
- 2010-09-16 WO PCT/JP2010/066005 patent/WO2011034115A1/fr not_active Ceased
- 2010-09-16 US US13/394,563 patent/US8685655B2/en active Active

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WO2013071918A1 (fr) *	2011-11-14	2013-05-23	Pluriselect Gmbh	Procede d'isolement de cellules et de bioparticules
WO2016120247A1 (fr) *	2015-01-26	2016-08-04	Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts	Méthodes de libération quantitative de peptides et de protéines biotinylés à partir de complexes de streptavidine
WO2020068547A1 (fr)	2018-09-25	2020-04-02	Siemens Healthcare Diagnostics Inc.	Procédés et compositions pour éliminer l'interférence de la biotine dans des dosages à l'aide de pièges moléculaires conjugués
EP3857226A4 (fr) *	2018-09-25	2021-12-15	Siemens Healthcare Diagnostics Inc.	Procédés et compositions pour éliminer l'interférence de la biotine dans des dosages à l'aide de pièges moléculaires conjugués
US11313855B2 (en)	2018-09-25	2022-04-26	Siemens Healthcare Diagnostics Inc.	Methods and compositions for removing biotin interference from assays using conjugated molecular traps
EP4325220A3 (fr) *	2018-09-25	2024-05-01	Siemens Healthcare Diagnostics Inc.	Procédés et compositions pour éliminer l'interférence de la biotine dans des dosages à l'aide de pièges moléculaires conjugués
US12099055B2 (en)	2018-09-25	2024-09-24	Siemens Healthcare Diagnostics Inc.	Methods and compositions for removing biotin interference from assays using conjugated molecular traps
WO2021158556A1 (fr) *	2020-02-03	2021-08-12	Illumina, Inc.	Composition de clivage de biotine-streptavidine et clivage de fragments de bibliothèque
CN114144523A (zh) *	2020-02-03	2022-03-04	因美纳有限公司	生物素-链霉抗生物素蛋白裂解组合物和文库片段裂解
US12146133B2 (en)	2020-02-03	2024-11-19	Illumina, Inc.	Biotin-streptavidin cleavage composition and library fragment cleavage

Also Published As

Publication number	Publication date
CN102575244B (zh)	2015-07-22
EP2479268B1 (fr)	2020-11-04
JPWO2011034115A1 (ja)	2013-02-14
CN102575244A (zh)	2012-07-11
US20120171763A1 (en)	2012-07-05
JP5686098B2 (ja)	2015-03-18
WO2011034115A1 (fr)	2011-03-24
EP2479268A4 (fr)	2014-03-12
US8685655B2 (en)	2014-04-01

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